Stimulation of hematopoiesis by ex vivo activated immune cells

ABSTRACT

A protocol of activating and administering human blood cells so that bone marrow histology and/or blood cell counts of patients suffering from a plastic anemia approach normal. The protocol includes culturing the blood cells in the presence of a cytokine and an ionophore. It is emphasized that this abstract is provided to comply with the rules requiring an abstract that will allow a searcher or other reader to quickly ascertain the subject matter of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. 37 C.F.R. § 1.72(b).

RELATED APPLICATIONS

This application is a division of application Ser. No. 10/159,148 filedMay 29, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to therapies for aplastic anemia, anemia andthrombocytopenic purpura. In particular, this invention relates to exvivo activated immune cells as therapies for aplastic anemia, anemia andthrombocytopenic purpura. Furthermore, the invention relates toapproaches to activate cells and corresponding cell culture approaches.

2. Background

Aplastic anemia is a disease characterized by ineffective hematopoiesis.Patients have varying degrees of abnormalities in production of allblood cell types. Although in most cases, the cause of the disease isunknown, radiation, benzene-based compounds, viruses (e.g., hepatitis),environmental toxins, and over the counter and prescription medicationshave been suspected to damage bone marrow, thereby leading to apoptosisof marrow stem cells. Regardless of the underlying causes, patients showsimilar clinical manifestations and disease progression courses.Aplastic anemia affects primarily young man and older persons of boththe genders. Annually, two to six per million worldwide develop thisdisorder, with a prevalence of incidences in the Orient as compared toEurope or the United States. Several causal phenomena are hypothesizedfor aplastic anemia: congenital, pregnancy, viral, and drugs andchemicals.

The most frequently cited causal agent of aplastic anemia is drugs orchemical exposure. Some agents, such as chloramphenicol, benzene,ionizing radiation, and antineoplastic agents, cause an aplasia that isdose-related in severity from person-to-person. In these cases, marrowrecovery usually occurs after withdrawal of the causal agent. Otheragents, including pesticides and some anticonvulsants andantimicrobials, cause a reaction which is not dose-related and,therefore, cannot be predicted with hematological monitoring duringadministration. During administration of drugs, aplasias may occur evenafter cessation of drug therapy. In contrast to patients with idiopathicaplastic anemia, those with drug or toxin exposure exhibit similarclinical and demographic characteristics, have a similar prognosis, anda more-or-less uniform response to therapy.

In the case of benzene-induced aplastic anemia, mild to moderate diseasesymptoms usually disappear after patients cease being exposed tobenzene. However, for patients with severe bone marrow failure or whocontinually need blood transfusions, effective and safe treatment hasnot often been heretofore available. To date, bone marrowtransplantation is the only known cure.

Mild aplastic patients are often treated with as little therapy aspossible. The rationale for minimum treatment for mildly aplasticpatients is to remove the causal agent, thereby enabling spontaneousrecovery. In young patients with severe anemia, bone marrowtransplantation with an HLA-matched donor is the treatment of choice.Bone marrow transplantation effects complete remission in nearly 80% ofcases. However, survival decreases to 10-20% when the donor andrecipient are mismatched at two or more loci. Complications associatedwith transplantation include graft rejection, acute or chronicgraft-versus-host disease, infection, and other miscellaneous organspecific damage. Marrow transplant recipients also have an increasedlong-term risk for developing subsequent solid tumors.

SUMMARY OF THE INVENTION

The present inventor has investigated using cultured (activated) bloodcells for treating patients with blood deficiencies, such as anemia,aplastic anemia and/or thrombocytopenic purpura. In one embodiment ofthe invention, a quantity of blood cells effective to treat blooddeficiencies when injected into a patient is provided. The quantity ofblood cells may be cultured in the presence of a cytokine and anionophore. The cytokine and ionophore may be present in effectiveconcentrations. The cytokine may comprise interleukin-2 andmacrophage-colony stimulating factor. The ionophore may comprise A23187.

In another embodiment, the present invention provides a process oftreating blood deficiencies in a patient, the treatment comprisingadministering ex vivo cultured blood cells to the patient. Atherapeutically effective amount of blood cells may be administered tothe patient. The blood cells may be autologous to the patient,allogeneic to the patient, or from an immunologically acceptable owner.The blood cells may further be cultured in the presence of a cytokineand an ionophore. The cytokine and ionophore may be present in effectiveamounts.

In yet another embodiment, the present invention provides a method ofculturing blood cells, the method comprising culturing the blood cellsin the presence of a cytokine and an ionophore. The blood cells may becultured in the presence, for example, of effective amounts of thecytokine and ionophore; may be cultured in a medium which may or may notcomprise mammalian serum; may be cultured for a period, for example,between about 2 and 200 hours or longer; and may be cultured at atemperature, for example, between about 30 and 42 degrees C. Thecytokine may include interleukin-2 and/or granulocyte macrophage-colonystimulating factor. The ionophore may include A23187.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows low-power views of H&E stained bone marrow biopsies fromthree patients responsive to the present therapy. The views labeled asA, C and E are marrows from patients before treatment. In these views,early empty and impaired marrows implicate severe aplastic anemia. Inthe views denoted as B, D and F, the marrows are from the same patientsafter treatment. These marrows show much improved distribution andcellularity.

FIG. 2 is a plot of platelet count in platelets per cubic millimeter asa function of days after starting treatment with activated cells for achild pateint with platelet deficiency.

DETAILED DESCRIPTION

The present invention includes a therapy of administering atherapeutically effect amount of ex vivo cultured blood cells topatients afflicted with blood deficiencies, such as anemia, aplasticanemia and thrombocytopenic purpura. The term “therapeutically effectiveamount” is intended to include a sufficient quantity of the presentactivated blood cells to effect a statistically significant increase inblood cell counts when administered to a patient with blooddeficiencies, i.e., a significantly low concentration of a natural bloodcomponent, such as red blood cells, white blood cells, platelets andother factors produced by the bone marrow and cells generated from thebone marrow. The cultured blood cells may be either from the patient orfrom an immunologically acceptable donor. One protocol for activatingblood cells via ex vivo culture includes obtaining a blood sample (e.g.,10-100 ml) from the patient, or an immunologically acceptable donor,separating blood cells from the blood sample, and culturing theseparated blood cells. An “immunologically acceptable donor” is a personhaving tissues, to include blood cells, that do not have medicallyunacceptable levels of recipient reactions (e.g., hemolytic anemia,heart failure, renal failure). The blood cells may be separated fromblood sera by protocols such as by centrifugation. The separated bloodcells are then cultured under sterile conditions in a medium with one ormore of a cytokine (to include cell stimulating factors) and anionophore. The separated blood cells may be cultured in the media asspecified above, for example, for periods between of greater than about1 hour, in other embodiments between about 10 and 200 hours, betweenabout 20 and 80 hours, or between about 30 and 60 hours and at atemperature, for example, between about 30 and 42 degrees C., in otherembodiments between about 32 and 40 degrees C., or between about 37 and38 degrees C. or any range subsumed therein. A person of ordinary skillin the art will recognize that other ranges of periods and temperatureswithin these explicit ranges are contemplated, and are within thepresent disclosure.

Blood deficiencies can be treated by the approaches described herein. Ingeneral, the blood deficiencies involve a reduced concentration of bloodcomponents that originate from the bone marrow or from products, such asspecific cell types, from the bone marrow. Blood deficiencies include,for example, anemia, aplastic anemia and thrombocytopenic purpura.Anemia can be considered broadly as a deficiency of a blood componentor, in some contexts, as a deficiency of red blood cells. Aplasticanemia is a deficiency of peripheral blood elements. Thrombocytopenicpurpura, such as idiopathic thrombocytopenic purpura, involves adeficiency in platelet number. As a specific example, the discussionbelow describes aplastic anemia in some detail, although the treatmentmethods can be applicable more broadly.

After being cultured, the activated blood cells may be washed (e.g.,twice with sterile saline solution). Therapeutically effective amountsof the activated blood cells are then administered to patients. Oneacceptable method of administering the activated blood cells isintravenously. While the activated cells may be administered in a singledose, portions of the activated blood cells may also be administeredover a period of time. For example, doses of the present activated bloodcells may be administered to patients once per week for a period of fourweeks. However doses of the present activated blood cells may beadministered to patients at intervals of, for example, one-half week,ten days, 14 days, 21 days, other intermediate periods, or othereffective periods. Moreover, the intervals may vary during the course ofthe treatment. For example, initially blood cell doses may beadministered at daily, twice a week, weekly, and/or bi-weekly intervals.The dosages can be, for example, between about 1×10⁵ to about 2×10⁸cells per treatment, which may depend on the patient's age andcondition. The total time required for treatment (e.g., administeringthe present activated blood cells) may depend on the amount of activatedblood cells available and patient response. Patient response can bemeasured, for example, in terms of return to normal blood cell countsand/or marrow histology as well as an overall improvement in health.Obviously, blood samples can be drawn from patients repeatedly during orafter the initial treatment period so that additional activated bloodcells can be obtained for further treatments. Furthermore, activatedblood cells from an immunologically acceptable donor can be administeredinitially or administered for the entire duration of the treatment.Alternatively, blood cells from the patient, activated by the presentprotocol, may be administered after blood cells from an immunologicallyacceptable donor are initially administered.

The most current definition of severe aplastic anemia is markedpancytopenia with at least two of the following: 1) granulocytes lessthan 500/microliter, 2) platelets less than 20,000/microliter, 3) anemiawith corrected reticulocyte count less than 1%, plus markedlyhypoplastic marrow depleted of hematopoietic cells. Moderate aplasticanemia generally involves a hypocellular bone marrow and cytopenia in atleast two cell lines not in the severe range. Onset is insidious and theinitial complaint may be progressive fatigue and weakness due to theanemia, followed in some cases by hemorrhage. The hemorrhage is usuallyfrom the skin and mucosal linings, due to thrombocytopenia. Infection israre despite the severe neutropenia. Physical examination reveals pallorand possibly bruising or petechiae. Aplastic anemia patients exhibit nolymphadenopathy or splenomegaly. Fever may or may not be present.Peripheral blood assays show pancytopenia. The presence of immature redand white blood cells strongly argues against aplastic anemia.

Red blood cells may be mildly macrocytic due to increased erythropoieticstress and they usually are normocytic and normochromic. The correctedreticulocyte count is very low or zero, indicating a lack oferythropoiesis. Bleeding time may be prolonged even with normalcoagulation parameters. Patients have an increased serum iron and anormal transferrin, resulting in an elevated transferrin saturation.Plasma iron clearance is decreased due to a reduction in erythropoiesis.Bone marrow aspirate may be dry. But a biopsy can show severehypocellular or aplastic marrow with fatty replacement. Because therehave been cases in which the initial marrow biopsy exhibitedhypercellularity, more than one biopsy may be necessary for accuratediagnosis. A severe depression can be noted in all hematopoieticprogenitor cells, including myeloid, erythroid, pluripotent cell lines,and megakaryocytes. Diagnosis generally is based on finding the classictriad of anemia, neutropenia, and thrombocytopenia in both blood andbone marrow specimens. X-rays may be needed to rule out bone lesions orneoplastic infiltrates. Magnetic resonance imaging has been useful inclearly defining hypoplastic marrow. Since the diagnosis is one ofexclusion, all other causes of pancytopenia and other lab findings areusually ruled out before aplastic anemia can be diagnosed.

The basic defect in aplastic anemia is failure of production of all celllines. Possible mechanisms of the pathogenesis of aplastic anemiainclude 1) defective or absent hematopoietic stem cells, 2) abnormalbone marrow microenvironment, 3) abnormal regulatory cells, and 4)suppression of hematopoiesis by immunologic cells.

While the pathophysiology of the disease is not yet completely clear,(Young et al., The pathophysiology of acquired aplastic anemia, N. Engl.J. Med. 1997; 336(19): 1365-1372 and Young et al., The treatment ofsevere acquired aplastic anemia, Blood. 1995; 85(12): 3367-3377) thereis evidence to support the theory that aplastic anemia is animmune-mediated disease. Bone marrow transplantation andimmunosuppressive therapy using combined antilymphocyte globulin andcyclosporine have been used for treatment (Rosenfeld et al., Intensiveimmunosuppression with antithymocyte globulin and cyclosporine astreatment for severe aplastic anemia, Blood 1995; 85(11): 3058-3065 andHalperin et al., Severe acquired aplastic anemia in children: 11-yearexperience with bone marrow transplantation and immunosuppressivetherapy, Am. J. Pediatr. Hematol. Oncol. 1989; 11(3): 304-309). However,the therapy of immune suppression often has undesirable and severe sideeffects. Moreover, hematopoietic growth factors such as granulocytecolony-stimulating factor (Kojima et al., Treatment of aplastic anemiain children with recombinant human granulocyte-colony stimulatingfactor, Blood 1991; 77(5): 937-941 and Sonoda et al., Multilineageresponse in aplastic anemia patients following long-term administrationof filgrastim (recombinant human granulocyte colony stimulating factor),Stem Cells 1993; 11: 543-554), granulocyte macrophage colony-stimulatingfactor (Champlin et al., Treatment of refactory aplastic anemia withrecombinant human granulocyte-macrophage-colony-stimulating factor,Blood 1989; 73(3): 694-699 and Guinan et al., A phase I/II trial ofrecombinant granulocyte-macrophage colony-stimulating factor forchildren with aplastic anemia, Blood 1990; 76(6): 1077-1082), andInterleukin-3 (Ganser et al., Effect of recombinant human interleukin-3in patients with normal hematopoiesis and in patients with bone marrowfailure, Blood 1990; 76(4): 666-676 and Nimer et al., A phase I/II studyof interluekin-3 in patients with aplastic anemia and myelodysplasia,Exp. Hematol. 1994; 22: 875-880) have provided only limited andtransient effects.

Many patients respond to immunosuppressive therapy and there areabnormal levels of various immune molecules in aplastic patients. Forinstance, Interleukin-1, produced by macrophages, natural killer cells,B lymphocytes, and endothelial cells, plays a central role in bothimmune responses and regulation of hematopoiesis by inducing the releaseof erythroid and multipotent colony-stimulating factors from marrowstromal cells, regulating early progenitor cells and stimulating stemcell recovery following induced myelosuppression. Immune dysregulationin aplastic anemia consists of decreased natural killer cell activity,increased numbers of activated T suppressor cells and abnormalproduction of Interleukin-2 and gamma-Interferon.

Natural killer cells are large granular lymphocytes which lyse tumorcells or virus-infected target cells upon direct contact. Natural killercells also produce gamma-interferon, Interleukin-2, and inducescolony-stimulating activity. These cells may inhibit myeloid anderythroid colony formation under certain conditions. For instance, whenexogenous growth factors are absent from a culture, natural killer cellsnormally produce cytokines and support hematopoiesis. However, optimalconditions induce natural killer cells to inhibit hematopoiesis. Naturalkiller cell activity in aplastic anemia patients returns to normal afterhematopoietic recovery.

Gamma-Interferon is produced by activated lymphocytes and suppresseshematopoiesis. Although aplastic patients show an overproduction ofgamma-Interferon, levels of gamma-Interferon decrease in response toimmunosuppression. Interferons are potent inhibitors of hematopoieticcolony formation—both through direct action on progenitor cells andindirect effects via accessory immune system cells.

Tumor necrosis factor-alpha is another cytokine which is in excess inaplastic anemia. It functions to inhibit colony growth of the normalhematologic progenitors. High tumor necrosis factor-alpha valuescorrelate with decreased platelet, hemoglobin, and leukocyte counts.Tumor necrosis factor-alpha and gamma-Interferon may act synergisticallyto suppressor hematopoiesis.

Aplastic anemia patients produce gamma-Interferon and tumor necrosisfactor-alpha in excess, show an inverted helper:suppressor T cell ratio,and have predominantly T suppressor cells in the bone marrow. Thesecells may mediate suppression of hematopoiesis via cytokine production.The bone marrow also has a higher proportion of cytotoxic T cells thanperipheral blood. The clinical relevance of immune dysfunction issuggested by a decrease in activated lymphocytes following successfulimmunosuppressive therapy.

Mechanisms for acquired aplastic anemia in general, and mechanisms forbenzene-induced aplastic anemia in particular, are not well understood.Nonetheless, both types of aplastic anemia share considerablesimilarities with respect to pathophysiology and clinicalmanifestations. There are presently two hypotheses to explain themechanism of aplastic anemia, direct damage and immune-mediated. Bothhypotheses are supported by data from experimental and clinical studies.Direct damage to bone marrow cells is thought to be responsible fortemporary and reversible bone marrow failure following cytotoxicchemotherapy and radiotherapy. Immune-mediated bone marrow failure ismore difficult to cure. In the case of benzene-induced aplastic anemia,the disease seems to be associated with both mechanisms. Evidence ofdirect damage to bone marrow cells is supported by the studiesindicating that benzene is involved in inhibiting a number ofbiochemical processes of bone marrow cells. Specifically, benzene hasbeen shown to damage stromal macrophages in bone marrow, thereby leadingto deficient interleukin-1 production (Niculescu et al., Inhibition ofthe conversion of pre-interleukins-1[alpha] and 1[beta] to maturecytokines by p-benzoquinone, a metabolite of benzene, Chemico-BiologicalInteractions; 1995; 98: 211-222 and Kalf et al., p-benzoquinone, areactive metabolite of benzene, prevents the processing ofpre-interleukins-1[alpha] and -1 [beta] to active cytokines byinhibition of the processing enzymes, calpain, and interluekin-1 [beta]converting enzyme, Environmental Health Perspectives; 1996; 104 (suppl.6): 1251-1256). Interleukin-1 is considered important for growth anddifferentiation of stem cells (Bagby, G. C., Production of multi lineagegrowth factors by hematopoietic stromal cells: an intercellularregulatory network involving mononuclear phagocytes and interleukin-1,Blood Cells 1987; 13:147-159 and Fibbe et al., Human fibroblasts producegranulocyte-CSF, macrophage-CSF and granulocyte-macrophage-CSF followingstimulation by interleukin-1 and poly(rl).poly(rC), Blood 1988; 72(3):860-866). However, there has been no report of prolonged response totreatments of hematopoietic growth factors, including interleukin-1.

Medium.

Suitable media used in ex vivo activation provide essential nutrientsfor blood cells. These media generally comprise, for example, inorganicsalts, amino acids, vitamins, and other compounds all in forms which canbe directly utilized by blood cells. By way of illustration and notlimitation, one suitable medium is RPMI 1640. However, other media, suchas serum-free media AIM-V, will support blood cells in culture may besuitable as well. The medium may be supplemented with a mammalian serum,e.g., fetal bovine serum at levels between about 0.1 and 50%, betweenabout 1 and 40%, or between about 5% and 15%, of the medium, by weight.One suitable formulation of RPMI, designated as a modified RPMI 1640 andavailable under catalog number 30-2001 from American Type CultureCollection, has the following ingredients: (g/liter) Inorganic SaltsCa(NO₃)₂.4H₂O 0.10000 MgSO₄ (anhydrous) 0.04884 KCl 0.40000 NaHCO₃1.50000 NaCl 6.00000 Na₂HPO₄ (anhydrous) 0.80000 Amino Acids L-Arginine(free base) 0.20000 L-Asparagine.H₂O 0.05682 L-Aspartic Acid 0.02000L-Cystine.2HCl 0.06520 L-Glutamic Acid 0.02000 L-Glutamine 0.30000Glycine 0.01000 L-Histidine (free base) 0.01500 Hydroxy-L-Proline0.02000 L-Isoleucine 0.05000 L-Leucine 0.05000 L-Lysine.HCl 0.04000L-Methionine 0.01500 L-Phenylalanine 0.01500 L-Proline 0.02000 L-Serine0.03000 L-Threonine 0.02000 L-Tryptophan 0.00500 L-Tyrosine.2Na.2H₂O0.02883 L-Valine 0.02000 Vitamins D-Biotin 0.00020 Choline Chloride0.00300 Folic Acid 0.00100 myo-Inositol 0.03500 Nicotinamide 0.00100p-Amino Benzoic Acid 0.00100 D-Pantothenic Acid 0.00025 (hemicalcium)Pyridoxine.HCl 0.00100 Riboflavin 0.00020 Thiamine.HCl 0.00100 VitaminB-12 0.000005 Other D-Glucose 4.50000 Glutathione (reduced) 0.00100HEPES 2.38300 Phenol Red, Sodium Salt 0.00500 Sodium Pyruvate 0.11000

-   1. Cytokines. One or more cytokines may be used to activate blood    cells when cultured in the presence thereof. Cytokines are small    proteins (usually in the range of 5-20 kD) that are released by    cells and have specific effects on cell-cell interaction,    communication, and behavior of other cells. Usually included as    cytokines, are interleukins, lymphokines and signaling molecules    such as tumor necrosis factor (TNF) and interferons. While natural    cytokines can be used, recombinant produced cytokines produced, for    example, by established nucleic acid expression systems are also    contemplated. As such, modified and mutated forms of natural    cytokines that maintain function can also be used. Exemplary    cytokines, which may be suitable for some embodiments of the present    invention, include:    -   A. Interleukins. A variety of naturally occurring polypeptides        that affect functions of specific cell types and are found in        small quantities. They are secreted regulatory proteins produced        by lymphocytes, monocytes and various other cells and are        released by cells in response to antigenic and non-antigenic        stimuli. The interleukins, of which there are 16 identified to        date, modulate inflammation and immunity by regulating growth,        mobility and differentiation of lymphoid and other cells.        Interleukins may be present in concentrations between about 10        and 50,000 IU/ml, about 100-5,000 IU/ml, or about 100-1,000        IU/ml. Alternatively an effective concentration of interleukins        may be present. An effective concentration of interleukins is        any concentration at which blood cells are actived by the        present protocol.        -   i. Interleukin-1 (IL-1). IL-1 is a soluble protein (17 kD:            152 amino acids) secreted by monocytes, macrophages or            accessory cells involved in the activation of both T            lymphocytes and B lymphocytes and potentiates their response            to antigens or mitogens. Biological effects of IL-1 include            the ability to replace macrophage requirements for T-cell            activation, as well as affecting a wide range of other cell            types. At least two IL-1 genes are known and alpha and beta            forms of IL-1 are recognized. IL-1 is released early in an            immune system response by monocytes and macrophages. It            stimulates T-cell proliferation and protein synthesis.            Another effect of IL-1 is to cause fever.        -   ii. Interleukin-2 (IL-2). IL-2 is a hormone-like substance            released by stimulated T lymphocytes. IL-2 causes activation            and differentiation of other T lymphocytes independently of            antigen. IL-2 stimulates the growth of certain            disease-fighting blood cells in the immune system and is            secreted by Thl CD4 cells to stimulate CD8 cytotoxic T            lymphocytes. IL-2 also increases the proliferation and            maturation of CD4 cells themselves.        -   iii. Interleukin-3 (IL-3). IL-3 is a product of mitogen            activated T-cells. IL-3 is a colony stimulating factor for            bone marrow stem cells and mast cells. IL-3 is considered            one of the hematopoietic colony stimulating factors.        -   iv. Interleukin-4 (IL-4). IL-4 is a soluble cytokine factor            produced by activated T lymphocytes that promotes antibody            production by causing proliferation and differentiation of            B-cells. IL-4 induces the expression of class II major            histocompatibility complex and fc receptors on B-cells. IL-4            also acts on T lymphocytes, mast cell lines, and several            other hematopoietic lineage cells including granulocyte,            megakaryocyte, and erythroid precursors, as well as            macrophages.        -   v. Interleukin-5 (IL-5). IL-5 is a factor promoting            eosinophil differentiation and activation in hematopoiesis.            It also triggers activated B-cells for a terminal            differentiation into Ig-secreting cells.        -   vi. Interleukin-6 (IL-6). IL-6 stimulates the growth and            differentiation of human B-cells and is also a growth factor            for hybridomas and plasmacytomas. It is produced by many            different cells including T-cells, monocytes, and            fibroblasts. IL-6 is a single chain 25 kD cytokine            originally described as a pre B-cell growth factor, now            known to have effects on a number of other cells including            T-cells which are also stimulated to proliferate.        -   vii. Interleukin-7 (IL-7). IL-7 is a hematopoietic growth            factor that promotes growth of B-cell precursors and is also            co-mitogenic with interleukin-2 for mature T-cell            activation. IL-7 is produced by bone marrow stromal cells.        -   viii. Interleukin-8 (IL-8). IL-8 is a cytokine that            activates neutrophils and attracts neutrophils and T            lymphocytes. IL-8 is released by several cell types            including monocytes, macrophages, T lymphocytes,            fibroblasts, endothelial cells, and keratinocytes by an            inflammatory stimulus. IL-8 is a member of the            beta-thromboglobulin superfamily and structurally related to            platelet factor 4.        -   ix. Interleukin-9 (IL-9). IL-9 is a cytokine produced by            T-cells, particularly when mitogen stimulated. IL-9            stimulates the proliferation of erythroid precursor cells            (BFUE) and is thought to be a regulator of hematopoiesis.            IL-9 may act synergistically with erythropoietin. The IL-9            receptor belongs to the hemopoietic receptor super family.            IL-9 has been shown to enhance the growth of human mast            cells and megakaryoblastic leukaemic cells as well as murine            helper T-cell clones. Il-9 is a glycoprotein that is derived            from T-cells and maps to human chromosome 5.        -   x. Interleukin-10 (IL-10). IL-10 is a factor produced by Th2            helper T-cells, some B-cells and LPS activated monocytes. It            is a coregulator of mast cell growth.        -   xi. Interleukin-11 (IL-11). IL-11 is a pleiotropic cytokine,            originally isolated from primate bone marrow stromal cell            line, that has the ability to modulate antigen-specific            antibody responses, potentiate megakaryocytes, and regulate            bone marrow adipogenesis. IL-11 stimulates T-cell dependent            B-cell maturation, megakaryopoiesis, and various stages of            myeloid differentiation.        -   xii. Interleukin-12 (IL-12). IL-12 is a 75 kD heterodimeric            cytokine composed of disulfide-bonded 40 kD and 35 kD            subunits that was originally identified by its ability to            induce cytotoxic effector cells in synergy with less than            optimal concentrations of interleukin-2. IL-12 is released            by macrophages in response to infection and promotes the            activation of cell-mediated immunity. Specifically, IL-12            triggers the maturation of Thl CD4 cells, specific cytotoxic            T lymphocyte responses, and an increase in the activity of            NK cells. Consequently, IL-12 is the initiator of            cell-mediated immunity. It enhances the lytic activity of NK            cells, induces interferon production, stimulates the            proliferation of activated T-cells and NK cells. Is secreted            by human B lymphoblastoid cells (NC 37).        -   xiii. Interleukin-13 (IL-13). IL-13 is a T            lymphocyte-derived cytokine that produces proliferation,            immunoglobulin isotype switching, and immunoglobulin            production by immature B-lymphocytes. IL-13 is produced by            activated T-cells, inhibits IL-6 production by monocytes,            and also inhibits the production of other pro-inflammatory            cytokines such as TNF, IL-1, and IL-8. IL-13 stimulates            B-cells. The gene for IL-13 is located on human chromosome            5q in a gene cluster that also has the IL-4 gene.        -   xiv. Interleukin-14 (IL-14). IL-14 is a cytokine that            induces B-cell proliferation, inhibits immunoglobulin            secretion, and selectively expands certain B-cell            subpopulations.        -   xv. Interleukin-15 (IL-15). IL-15 is a cytokine that            stimulates the proliferation of T lymphocytes and shares            biological activities with IL-2. Il-15 also can induce B            lymphocyte proliferation and differentiation.        -   xvi. Interleukin-16 (IL-16). IL-16 is a cytokine produced by            activated T lymphocytes that stimulates the migration of            CD4-positive lymphocytes and monocytes.    -   B. Lymphokines. A lymphokine is a substance produced by a        leucocyte that acts upon another cell. Examples are        interleukins, interferon alpha, lymphotoxin (tumor necrosis        factor alpha), granulocyte monocyte colony stimulating factor        (GM-CSF).        -   i. Interferons (IFN) are a family of glycoproteins human            cells which normally have a role in fighting viral            infections by preventing virus multiplication in cells.            Interferons may be present in the same concentrations as            interluekins. Alternatively, effective concentrations of            interferons may be present. Effective concentrations of            interferons are contemplated to include any concentration at            which blood cells are activated by the present protocol. IFN            alpha is secreted by leucocytes and IFN gamma is secreted by            fibroblasts after viral infection.            -   1. Interferon gamma is an interferon elaborated by T                lymphocytes in response to either specific antigen or                mitogenic stimulation.            -   2. Interferon alpha includes a number of different                subtypes that are elaborated by leukocytes in response                to viral infection or stimulation with double-stranded                RNA. IFN-alpha-2A and -2B are protein products made by                recombinant DNA techniques and are used as                antineoplastic agents. Interferon-alpha is one of the                type I interferons (interferon type I) produced by                peripheral blood leukocytes or lymphoblastoid cells when                exposed to live or inactivated virus, double-stranded                RNA, or bacterial products. It is the major interferon                produced by virus-induced leukocyte cultures and, in                addition to its pronounced antiviral activity, causes                activation of natural killer cells.            -   3. Interferon alfa-2a is a type I interferon consisting                of 165 amino acid residues with lysine in position 23.                This protein is produced by recombinant DNA technology                and resembles interferon secreted by leukocytes. It is                used extensively as an antiviral or antineoplastic                agent.            -   4. Interferon alfa-2b is type I interferon consisting of                165 amino acid residues with arginine in position 23.                This protein is produced by recombinant DNA technology                and resembles interferon secreted by leukocytes. It is                used extensively as an antiviral or antineoplastic                agent.            -   5. Interferon beta is an interferon elaborated by                fibroblasts in response to the same stimuli as                interferon alpha. Interferon-beta is one of the type I                interferons produced by fibroblasts in response to                stimulation by live or inactivated virus or by                double-stranded RNA. It is a cytokine with antiviral,                antiproliferative, and immunomodulating activity.            -   6. Interferon-b2 (interleukin-6) is a cytokine that                stimulates the growth and differentiation of human                B-cells and is also a growth factor for hybridomas and                plasmacytomas. It is produced by many different cells                including T-cells, monocytes, and fibroblasts. INF-b2 is                a single chain 25 kD cytokine originally described as a                pre B-cell growth factor, now known to have effects on a                number of other cells including T-cells, which are also                stimulated to proliferate. INF-b2 is an inducer of acute                phase proteins and a colony stimulating factor acting on                mouse bone marrow.            -   7. Interferon gamma is elaborated by T lymphocytes in                response to either specific antigen or mitogenic                stimulation.        -   ii. Tumor necrosis factor (TNF) is a tumor-inhibiting factor            present in the blood of animals exposed to bacterial            lipopolysaccharide. TNF preferentially kills tumor cells in            vivo and in vitro, causes necrosis of certain transplanted            tumors in mice, and inhibits experimental metastases. Human            TNF alpha is a protein of 157 amino acids and has a wide            range of pro-inflammatory actions. TNF may be present in the            same concentrations as interleukins. Alternatively, TNF may            be present in an effective concentration. An effective            concentration of TNF is an concentration at which blood            cells are activated by the present protocol.    -   C. Cell Stimulating Factors. Activating blood cells in the        presence of one or more cell stimulation factors may be        efficacious in alleviating aplastic anemia in the context of the        present invention. Cell stimulating factors are contemplated to        include such substances as granulocyte colony-stimulating factor        granulocyte macrophage-colony stimulating factor and        macrophage-colony stimulating factor. Cell stimulating factors        may be present in concentrations between about 10 and 50,000        IU/ml, between about 10 and 10,000 IU/ml, or between about 10        and 1000 IU/ml. Alternatively, an effective concentration of        cell stimulating factors may be present. An effective        concentration of cell stimulating factors is any concentration        at which blood cells are activated by the present protocol.        -   1. Granulocyte colony-stimulating factor (G-CSF): G-CSF are            glycoproteins synthesized by a variety of cells and are            involved in growth and differentiation of hematopoietic stem            cells. In addition, these factors stimulate the end-cell            functional activity of stem cells.        -   2. Granulocyte-macrophage colony-stimulating factor            (GM-CSF): GM-CSF is an acidic glycoprotein of 23 kD with            internal disulfide bonds. GM-CSF is produced in response to            a number of inflammatory mediators by mesenchymal cells            present in the hemopoietic environment and at peripheral            sites of inflammation. GM-CSF stimulates the production of            neutrophilic granulocytes, macrophages, and mixed            granulocyte-macrophage colonies from bone marrow cells and            can stimulate the formation of eosinophil colonies from            fetal liver progenitor cells.        -   3. Macrophage-colony stimulating factor (M-CSF): M-CSF is a            cytokine synthesised by mesenchymal cells that stimulates            pluripotent stem cells of bone marrow into differentiating            towards the production of monocytes (mononuclear            phagocytes). The compound stimulates the survival,            proliferation, and differentiation of hematopoietic cells of            the monocyte-macrophage series. It is a disulfide-bonded            glycoprotein dimer with a mw of 70 kD and binds to a single            class of high affinity receptor which is identical to the            product of the c-fms proto-oncogene.-   2. Ionophores. Ionophores are calcium or other cation specific    reagents (such as polypeptrates) which can traverse a lipid bilayer    and a lipid soluble. There are two classes of ionophores: carriers    and channel formers. Carriers, like valinomycin, form cage-like    structures around specific ions, diffusing freely through the    hydrophobic regions of the bilayer. Channel formers, like    gramicidin, form continuous aqueous pores through the bilayer,    allowing ions to defuse therethrough. In addition to the foregoing,    suitable ionophores for the present protocol may include A23187    (calcimycin), ionomycin, geldanamycin, monensin (Na-salt), nystatin,    polymyxin-B sulfate, and rapamycin. It is believed that carriers,    such as A23187, accumulate calcium cations in response to pH    gradients. A23187 possesses a dissociating carboxylic acid group and    catalyzes an electrically neutral exchange of protons for other    cations across the membrane (Hyono et al., BBA 389, 34-46 (1985):    Kolber and Haynes, Biophysics Journal, 36, 369-391 (1981); Hunt and    Jones, Biosci. Rep., 2, 921-928 (1982)). Two molecules of A23187 are    present as carboxylate anions, and are thus available to carry to    protons, or equivalents, back across the membrane after releasing    the transported divalent cation. If present, ionophores may be    present in concentrations between about 1 and 10,000 ng/ml, between    about 1 and 1000 ng/ml, or between about 10 and 500 ng/ml.    Alternately, ionophores may be present in an effective    concentration. An effective concentration of ionophores is any    concentration at which blood cells are activated, but not    overactivated, by the present protocol. Excessive concentrations of    activating agents may not be effective in the treatment approaches    described herein.    Use, Packaging and Distribution

The delivery of activated cells can provide a statistically significantimprovement in clinical parameters of a patient. For example, theadministration of cell activated as described herein can result in astatistically significant increase in white blood cell counts, red bloodcell counts hemoglobin levels and platelet counts. In general,continuation of the treatment procedure as described herein can resultin a return to normal blood levels. In some embodiments, after fourtreatments, the patient can have an increase in each of white blood cellcounts, red blood cell counts and hemoglobin of at least about 20%, inother embodiments at least about 35% and in other embodiments at leastabout 50%. Similarly, in some embodiments, platelet counts can increaseby at least about 25%, in other embodiments at least about 50%, and infurther embodiments at least about 100%. A person of ordinary skill inthe art will recognize that additional ranges of blood parameterimprovement within the explicit ranges presented are contemplated andare within the present disclosure.

The activation compounds, such as one or more cytokines and/or one ormore ionophore, can be mixed with an appropriate cell culture medium ora portion thereof for distribution. In alternative embodiments, one ormore activation compounds can be packaged along with a cell culturemedium or portions thereof for shipping. Similarly, a desiredcombination of activation compounds, such as one or more cytokines andone or more ionopores, can be packaged together for shipping, eithermixed or in separate compartments. In any of these embodiments, themedium and/or activation compounds can be combined with any remainingmedium components and/or activation compounds to form the desired mediumfor culturing cells under conditions to activate the cells. Also, in anyof these embodiments, the compositions that are packaged together caninclude, for example, instructions for completing the cell culturemedium with activation properties and/or instructions for performing thecell culturing.

The cell culturing can be performed at the facility that is treating thepatient or the cell culturing to activate the cells can be performed ata remote location. In either case, the activated cells can beadministered after a short period of time after harvesting from the cellculture to ensure that the cells remain viable. Alternatively, the cellscan be stored under conditions that maintain the cells in a viablecondition. For example, the cells can be stored at liquid nitrogentemperatures with a cryoprotectant. The cells can be prepared, forexample using known procedures, at appropriate times for administrationto the patient. For example, the cells can be suspended in a bufferedsaline solution for administration to the patient. Other known carriers,for example, can be used for delivery of the cells.

EXAMPLES Example 1 Treatment of Aplastic Anemia

I. Patients

Eight patients with verified histories of from one to six years ofoccupational exposure to benzene were subjected to the present regimenafter their consents were obtained. The makeup of the patients was onemale and seven females and the ages of the patients ranged from 24 to41. All patients experienced symptoms of weakness, dizziness, fainting,and accelerated heart rates. Among these patients, four werehospitalized due to acute symptoms with bleeding. The hospitalizedpatients required blood or platelet transfusions. The other fourpatients experienced chronic symptoms and were treated with standardtherapies for four, six and 15 months, respectively. Bone marrowbiopsies and aspiration samples were obtained from all patients toconfirm hematopoiesis. Toxic levels of benzene were present in the bloodand bone marrow of all patients.

II. Purification of Peripheral Blood Mononuclear Cells and Cell Culture

Peripheral blood mononuclear cells (PBMCs) were separated from patientblood samples (40-50 ml) by Ficoll-Hypaque centrifugation. The separatedPBMCs were then placed in an appropriate volume (based on cellconcentration) of RPMI 1640 with 10% fetal bovine serum under sterileconditions and cultured at 2×10⁶ cells/ml for 48 hours in the presenceof interleukin-2 (IL-2) at 500 IU/ml (Chiron, Emeryville, Calif.),granulocyte macrophage-colony-stimulating factor (GM-CSF) at 200 IU/ml(Immunex, Seattle, Wash.), and calcium ionophore A23187 at 100 ng/ml(Sigma, St. Louis, Mo.). At the end of the culture period, adherentcells were scraped off the plastic surfaces of the culture vessels andharvested together with non-adherent cells. To harvest the cells, thecells were spun down to form a cell pellet. Different numbers of cellswere obtained for different patients. The harvested cells were washedtwice in saline solution and administered to the patients. Afterwashing, the cells were resuspended in 5 to 10 mls of saline, with thevolume determined by the number of cells. These suspensions were furtherdiluted with 50 ml of saline before administering the cells to thepatients.

Treatment Protocol

Activated allogeneic PBMCs were used for a single patient (HC) in thefirst three treatments because the patient had experienced low bloodcounts, severe bleeding and infection. For the other patients, activatedPBMCs were intravenously administered with 50 ml saline to the patients.The treatment was repeated every week for at least four weeks. Thenumber of cells administered to a particular patient depended on thenumber of cells obtained from the patient.

III. Results

Hematological Parameters

Hematological parameters, white blood cell counts, red blood cellcounts, hemoglobin levels, and platelet counts, were monitored beforeand after the treatment for each patient and are shown in Table 1. Datafrom these patients indicated that the therapy was effective inenhancing the peripheral blood cell counts. Six patients experiencedimprovement of more than one subset listed and two patients had betterplatelet counts. The blood cell counts began to improve in most patientsafter two treatments and continued to improve throughout the time thepresent activated cells were administered. Seven of the eight patientsimproved to the extent that some of their hemological parameters reachnormal levels or levels approaching normal after completion of fourtreatments. Although blood cell counts of the patients improved from thetherapy in general, improvements were not uniformly achieved. Somepatients experienced limited improvement in red blood cell counts, butdramatic improvement in platelet counts. It was noted that all patients'platelet counts were significantly increased.

Patient HC experienced more severe acute symptoms than the otherpatients. Additionally, patient HC had a bleeding problem as well.Because of the low yields of peripheral blood cells from patient HC,allogeneic PBMCs were used to stimulate patient HC's hematopoiesis.After three treatments using allogeneic cells, patient HC's blood countsbegan to improve. After the three treatments of allogeneic cells,autologous PBMCs were then used to continue the therapy. Althoughpatient HC's hemotological parameters were not corrected to normallevels after six treatments, patient HC continued to improve.

Discomfort due administering the present immunotherapy was mild tomoderate. Five patients experienced no appreciable discomfort. Threepatients experienced chilling, fevers between 37 and 39 degrees C.,headaches, nausea, vomiting, and loss of appetite after cell infusion.However, these symptoms were transient, typically lasting one to twodays. Aspirin was administered when patients experienced discomfort.

Bone Marrow Hematopoiesis

Bone marrow biopsies and aspiration samples were obtained from allpatients before the therapy began and two weeks after the finaltreatment. As shown in FIG. 1, the histology of the bone marrow samplesfrom three patients with the most severe samples indicated severe damagebefore the therapy was begun. After the therapy was administered,remarkable improvements in bone marrow histology were found. Withrespect to patient HC, however, the improvement observed in patient HC'sbone marrow was not coupled with improved peripheral blood counts.

Blood Transfusion

Before beginning treatment, four of the eight patients experiencedsevere symptoms, coupled with bleeding. These four patients requiredperiodic transfusions of whole blood or platelets before and during thetherapy. After four treatments, however, none of the patientsexperienced bleeding and whole blood and platelet transfusions were notcontinued.

Duration

The beneficial effects of the present cell-based therapy do not appearto be transient. All patients continued to have improved or stablehematological parameters after the therapy was discontinued. Some femalepatients experienced unstable blood counts during menstrual periods, butno patients experienced a relapse. Patient LC, who responded to thetherapy, has experienced stable symptoms for more than two months sincethe final treatment (FIG. 2).

Discussion

The results of this study suggest that administering activated PBMCs topatients with aplastic anemia is highly effective.

Some patients had close to normal bone marrow histologically, but hadperipheral hematological parameters which were not as close to normal.To this end, it seemed that a time gap occurred between histologicalrecovery of bone marrow and recovery of peripheral blood cell counts.Patients experiencing this gap were closely monitored and the patients'hematological parameters showed continued improvement. These patientssometimes took a few weeks or months to attain normal peripheral bloodcell counts.

In analyzing the data generated by the study, it was noted that, amongdifferent compartments of the blood, increase in platelets was mostevident, significant and rapid in patients benefiting from therapy. Theinitial increase in platelet counts was possibly due to the fact thatplatelets have a faster generation and differentiation interval. Othercell types of blood such as neutrophils, granulocytes and reticulocyteswere also improved in agreement with the four parameters listed (datanot shown). Platelet counts are likely more susceptible to benzenetoxicity than other blood cells, but are the most responsive to thepresent therapy due to their faster generation interval.

Acquired aplastic anemia is a difficult disease to cure. However, thepresent immunosuppression therapy was very effective in treating thisdisease, for which bone marrow transplants are the only known cureheretofore. However, in spite of the success of bone marrow transplants,this therapy has serious complications, e.g., tumors, (Socie et al.,Malignant tumors occurring after treatment of aplastic anemia, N. Eng.J. Med. 1993; 329(16): 1152-1157) and graft-versus-host disease (Ferraraet al., Graft-versus-host disease, N. Engl. J. Med. 1991(324); 324:667-674). Moreover, many patients cannot obtain bone marrow transplantsdue to the expense of the procedure and/or the lack of compatibledonors. To this end, a simple and effective therapy with fewer sideeffects is needed to treat aplastic anemia. The results of this studyindicate that aplastic anemia can be effectively treated with minimalside effects. The present cell-based immunotherapy is believed to beapplicable to other types of anemia and bone marrow disorders as well.These disorders include those experienced by HIV (human immunodeficiencyvirus)-infected patients after cocktail chemotherapy and cancer patientswith bone marrow failure after chemotherapy and radiotherapy, inheritedaplastic anemia, and idiopathic thrombocytopenic purpura.

While not wishing to be bound by a specific theoretical basis for theoperation of this invention, it is presently believed that severalphenomena may be responsible for the favorable responses of patients tothe present immunotherapy. A first theory is that the activated cellssecrete multiple (perhaps partially unknown) effective factorssimultaneously. These multiple factors, when working in concert, mayhave a synergistic combined effect. A second factor hypothesized for theeffectiveness of the present therapy is that some presently unknown keyfactors for hematopoiesis are produced by activated immune cells. Theseunknown factors may be responsible, at least in part, for theeffectiveness of the present therapy. A third factor which might beinvolved is that immune cells are capable of traveling to bone marrowand of delivering cytokines to hematopoietic stem cells and to otherprecursor cells at close range. Moreover, the present activated immunecells may be able to remain in close proximity to the marrow for periodssufficient to effect microenvironment improvement in the bone marrow. Afourth factor which might be responsible for the effectiveness of thepresent therapy is that cell contact between immune cells andhematopoietic cells may be essential for hematopoietic cell growth anddifferentiation. A fifth factor might be that activated immune cells,even in small amounts, may contribute to prevent the immune system fromadversely influencing hematopoiesis. Quantities of PCMBs from 10-100 mlof blood are relatively small. However, these small quantities exertedlarge effects on bone marrow histology and hematopoiesis.

The results of administering blood cells activated by the presentprotocol are unexpected in view of results from previous studies. Withthe exception of one study, Young et al. (note 2) found administeredgrowth factors (granulocyte-colony stimulating factor and granulocytemacrophage-colony stimulating factor) to affect neutrophil numbers only.The one study showed marked increases of neutrophil and platelet countswhen granulocyte-colony stimulating factor was administered.Interleukin-3, administered alone or in combination with granulocytemacrophage stimulating factor had even less effect on myelopoiesis thanthe growth factors administered alone. Similarly (Liu et al., Cellularinteractions in hemopoiesis, Blood Cells 1987; 13: 101-110 andEttinghausen et al., Hematologic effects if immunotherapy withlymphokine-activated killer cells and recombinant interleukin-2 incancer patients, Blood 1987; 69(6): 1654-1660), found that administeringactivated pheipheral blood mononuclear cells and interleukin-2 topatients “emphasized” anemia and oesinophilia in patients receiving thistherapy.

The present invention is also contemplated to include items ofmanufacture, which include separately packaged containers of one or morecytokine(s) and ionophore(s) as more fully described above. Thecontainer contents may be used to culture, and thereby activate, bloodcells for use in the present therapeutic protocol. Instructions, such ason a label, may be present in the item of manufacture. A medium suitablefor culturing blood cells may further be included.

Example 2 Treatment of Platelet Deficiency

This example described the treatment of a 1 year five month old femalepatient with idiopathic thrombocytopenic purpura. The patient wasdiagnosed with the disease at about 9 months.

The patient was first treated with conventional therapy ofcorticosteroids and intravenous infusiions of immunoglobulin. Althoughthe patient esponded to the conventional treatment, the patient becamecompletely dependent on the corticosteroid therapy. The maintainsufficient platelet levels, the patient had to receive increasinglyhigher doses of corticosteroids.

Then, the patient was treated with an activated cell based therapy asdescribed herein. The treatment was the same as described in Example 1except that only 20 mls of blood was drawn from the patient each time,rather than 40-50 mls. The patient was treated once a week for 9 weeks.Ex vivo activated cells were administered on day 1, day 8, day 15, day22, day 29, day 35, day 42, day 49 and day 56. At the same time thatimmunotherapy with activated cells was initiated, corticosteroids andany other aspect of conventional therapy were completely withdrawn. Thepatient's platelet levels gradually improved during the treatment withactivated cells as shown in FIG. 2. The patient had a lung infection atday 49 that correlated with a significant decrease in platelet number.After the patient recovered from the infection, the patient's plateletnumbers went back to normal levels.

All publications, patents, patent applications, and other documentscited herein are hereby incorporated by reference in their entirety. Inthe case of conflict, the present specification shall prevail.

Because numerous modifications of this invention may be made withoutdeparting from the spirit thereof, the scope of the invention is not tobe limited to the embodiments illustrated and described. Rather, thescope of the invention is to be determined by the appended claims andtheir equivalents. TABLE 1 Hematological Profiles of Patients Before andAfter Cell-Based Immune Therapy. No. of Age/ Treat- Disease WBC RBC HGBPLT WBC RBC HGB PLT Patients Sex ment Type (×10³/mL) (×10⁶/mL) (g/dl)(×10³/mm³) (×10³/mL) (×10⁶/mL) (g/dl) (×10³/mm³) HC 32/F 6 Acute 2.7 +/−0.2 1.2 +/− 0.1  4.0 +/− 0.3  28 +/− 3 3.8 +/− 0.25 2.0 +/− 0.2  7.0 +/−0.3  43 +/− 3 YM 29/F 4 Chronic 3.6 +/− 0.3 3.8 +/− 0.5 11.1 +/− 0.4  99+/− 11 6.7 +/− 0.3 3.9 +/− 0.4 12.7 +/− 0.5 182 +/− 17 TB 33/F 4 Acute3.2 +/− 0.2 2.3 +/− 0.3  8.4 +/− 0.4  47 +/− 5 4.7 +/− 0.3 3.2 +/− 0.311.5 +/− 0.7 107 +/− 11 LC 25/M 4 Acute 1.4 +/− 0.2 1.6 +/− 0.3  5.7 +/−0.3  16 +/− 7 5.7 +/− 0.2 4.8 +/− 0.3 13.5 +/− 0.7 135 +/− 14 YX 25/F 4Chronic 2.5 +/− 0.3 2.7 +/− 0.2  8.2 +/+ 0.4  22 +/− 3 3.5 +/− 0.3 2.8+/− 0.2  9.8 +/− 0.5 100 +/− 10 JX 29/F 5 Acute 2.7 +/− 0.7 3.0 +/− 0.2 8.1 +/− 3.4  44 +/− 18 4.5 +/− 0.7 3.6 +/− 0.1 12.0 +/− 0.4 126 +/− 8ZL 41/F 4 Chronic 3.1 +/− 0.6 4.0 +/− 0.6 12.6 +/− 1.9 154 +/− 35 3.7+/− 0.1 4.3 +/− 0.02 13.2 +/− 1.3 187 +/− 21 SC 29/F 4 Chronic 2.4 +/−0.3 2.6 +/− 0.1 10.0 +/− 3.5  54 +/− 4 2.8 +/− 0.2 2.9 +/− 0.2 11.0 +/−0.3  71 +/− 8The hematological parameters were measured and analyzed for fiveconsecutive days before and after the therapy.Data are expressed as means +/− standard deviation.WBC indicates white blood cells;RBC indicates red blood cells;HGB indicates hemoglobin;PLT indicates platelets.

1. A method of culturing blood cells, comprising culturing the bloodcells in the presence of an ionophore and between about 10 and about50,000 IU/ml of a cytokine.
 2. The method of claim 1, in which the bloodcells are cultured in the presence of effective amounts of the cytokineand the ionophore.
 3. The method of claim 1, in which the blood cellsare cultured in a medium comprising mammalian serum.
 4. The method ofclaim 1, in which the blood cells are cultured for a period betweenabout 10 and 90 hours.
 5. The method of claim 1, in which the bloodcells are cultured at a temperature between about 30 and 42 degrees C.6. The method of claim 1, in which the blood cells are cultured in thepresence of interleukin-2 and granulocyte macrophage-colony stimulatingfactor.
 7. The method of claim 1, in which the blood cells areallogeneic to a patient.
 8. The method of claim 1, in which the bloodcells are from an immunologically acceptable owner.
 9. The method ofclaim 1, in which the blood cells are autologous to a patient.
 10. Themethod of claim 1, in which the blood cells are cultured in the presenceof A23187.
 11. The method of claim 1, in which the blood cells are atleast partially separated from sera before being cultured.
 12. Themethod of claim 1, in which the blood cells are at least partiallyseparated from sera by being centrifuged before being cultured.
 13. Themethod of claim 1, in which the blood cells are at least partiallyseparated from blood by Ficoll-Hypaque centrifugation of the blood. 14.The method of claim 1, further comprising culturing with a culturemedium having at least one amino acid.
 15. The method of claim 1,wherein the ionophore is present in a concentration of between about 1and about 10,000 ng/ml.
 16. The method of claim 1, wherein the ionophoreis present in a concentration of between about 10 and about 500 ng/ml.17. The method of claim 1, wherein the cytokine is present in aconcentration of between about 100 and about 5,000 IU/ml.
 18. A quantityof activated blood cells effective to treat blood deficiencies followinginjection into a patient.
 19. The quantity of blood cells of claim 18,in which the quantity of blood cells is cultured in the presence of acytokine and an ionophore.
 20. The quantity of blood cells of claim 19,in which the quantity of blood cells is cultured in the presence ofbetween about 10 and about 50,000 IU/ml of the cytokine.
 21. Thequantity of blood cells of claim 19, in which the ionophore is presentin a concentration of between about 1 and about 10,000 ng/ml.
 22. Thequantity of blood cells of claim 19, in which the quantity of bloodcells is cultured in the presence of effective concentrations of thecytokine and ionophore.
 23. The quantity of blood cells of claim 18, inwhich a therapeutically effective amount of the cultured blood cellselevates peripheral blood cell populations in the patients whenadministered to the patients.
 24. The quantity of blood cells of claim18, in which the quantity of blood cells is cultured in the presence ofan interleukin and an ionophore.
 25. The quantity of blood cells ofclaim 18, in which the quantity of blood cells is cultured in thepresence of interleukin-2, macrophage-colony stimulating factor, and acalcium ionophore.
 26. The quantity of blood cells of claim 18, in whichthe quantity of blood cells is cultured in the presence ofinterleukin-2, granulocyte macrophage-colony stimulating factor, andA23187.
 27. A blood cell culture, comprising blood cells, an ionophore,and a concentration of between about 10 and about 50,000 IU/ml of acytokine.
 28. The blood cell culture of claim 27, wherein the bloodcells are present in a therapeutically effective amount.
 29. The bloodcell culture of claim 27, further comprising mammalian serum.
 30. Theblood cell culture of claim 27, wherein the culture has a temperaturebetween about 30 and about 42 degrees C.
 31. The blood cell culture ofclaim 27, wherein the medium further comprises interleukin-2 andgranulocyte macrophage-colony stimulating factor.
 32. The blood cellculture of claim 27, wherein the blood cells are allogeneic relative toa patient.
 33. The blood cell culture of claim 27, wherein the bloodcells are immunologically acceptable relative to a patient.
 34. Theblood cell culture of claim 27, wherein the blood cells are autologousrelative to a patient.
 35. The blood cell culture of claim 27, whereinthe ionophore comprises A23187.
 36. The blood cell culture of claim 27,wherein the blood cells are at least partially free of sera of thesource blood of the blood cells.
 37. The blood cell culture of claim 27,wherein the blood cells are of a type derived from centrifuged sera. 38.The blood cell culture of claim 27, wherein the blood cells are of atype derived from Ficoll-Hypaque centrifugation of blood.
 39. The bloodcell culture of claim 27, further comprising a culture medium having atleast one amino acid.
 40. The blood cell culture of claim 27, whereinthe ionophore is present in a concentration of between about 1 and about10,000 ng/ml.
 41. The blood cell culture of claim 27, wherein theionophore is present in a concentration of between about 10 and about500 ng/ml.
 42. The blood cell culture of claim 27, wherein the cytokineis present in a concentration of between about 100 and about 5,000IU/ml.